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Experimental Elaphostrongylus cervi Infection in Sheep and Goats
J. Comp. Path. 2000, Vol. 123, 248–257 doi:10.1053/jcpa.2000.0414, available online at http://www.idealibrary.com on
Experimental Elaphostrongylus cervi Infection in Sheep and Goats K. Handeland, L. M. Gibbons∗ and A. Skorping† Section of Wildlife Diseases, National Veterinary Institute, P.O. Box 8156 Dep., N-0033 Oslo, Norway, ∗Department of Pathology and Infectious Diseases, The Royal Veterinary College, University of London, Hawkshead Lane, North Mymms, Hatfield, Herts AL9 7TA, UK and †Department of Zoology, University of Bergen, Allegt. 41, 5007 Bergen, Norway Summary The pathogenesis and migratory life cycle of Elaphostrongylus cervi were studied in four sheep and six goats killed and examined 6 days to 5 months after inoculation with infective third-stage larvae (L3). Detailed histological studies demonstrated that the L3 followed a porto-hepatic, and probably also a secondary lymphatic, migratory route from the abomasum and small intestine to the lungs, with subsequent spread via the general circulation to the central nervous system (CNS) and other tissues. In addition, the results suggested that haematogenously spread L3, arrested in arterial vessels outside the spinal cord, migrated into the cord along the spinal nerves. During migration, the L3 caused focal inflammation and necrosis in the organs and along the spinal nerve roots, and infarcts occurred in the myocardium, kidneys and CNS. Nematode development took place in the CNS. During development, there was a gradual die-off of nematodes and patent infections were not observed. However, in one animal many mature nematodes were demonstrated in the CNS. In the nervous system, the nematodes caused encephalomyelitis, focal traumatic encephalomalacia, gliosis, meningitis, choroiditis, radiculitis and perineuritis. Two goats and one sheep displayed long-lasting paraparesis starting 6 weeks after inoculation. The signs apparently resulted from nematode-induced spinal nerve root lesions. From 19 weeks after inoculation the sheep also showed signs of severe brain disturbances due to traumatic and inflammatory lesions caused by adult E. cervi in the cerebral parenchyma. We conclude that E. cervi represents a potential cause of neurological disease in small ruminants grazing areas inhabited by red deer. This is the first report confirming the infectivity of E. cervi for domestic ruminants. 2000 Harcourt Publishers Ltd
Introduction In their definitive hosts the protostrongylid nematodes Elaphostrongylus rangiferi of reindeer (Rangifer tarandus) and E. cervi of red deer (Cervus elaphus) develop to the adult stage in the central nervous system (CNS) and then migrate to the skeletal muscles (Hemmingsen et al., 1993; Handeland, 1994; Handeland et al., 2000). The hosts may develop neurological signs during the CNS phase of the infection (Roneus and Nordkvist, 1962; Borg, 1979; Handeland et al., 1994). Mature females deposit eggs into veins and the eggs are carried haematogenously to the lungs (Prosl and Kutzer, 1980). Patent infections are characterized by the occurrence of nematode eggs disseminated throughout the lung tissue (Prosl and Kutzer, 1980) 0021–9975/00/080248+10 $35.00
and the presence of dorsal-spined first-stage larvae (L1) in the bronchial mucus and faeces (Mitskevich, 1964). The free-living L1 develop to the infective third-stage (L3) in various intermediate snail hosts (Skorping and Halvorsen, 1980) with definitive hosts becoming infected through accidental ingestion of snails containing L3, mainly during late summer (Mitskevich, 1964; Panin, 1964). Elaphostrongylus rangiferi is known to infect sheep and goats grazing in areas with reindeer, causing neurological disease (Handeland, 1991; Handeland and Sparboe, 1991; Handeland and Slettbakk, 1995). Signs are normally associated with the migration of adult nematodes in the CNS parenchyma. Experimental studies have shown that there is a gradual die-off of the parasite during its development in the CNS of small ruminants and 2000 Harcourt Publishers Ltd
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the parasite does not seem to reach patency in these unnatural hosts (Handeland and Skorping, 1992b, 1993; Handeland et al., 1993). Although infection with E. cervi has not been confirmed in domestic livestock, this nematode is considered a plausible cause of cerebrospinal nematodosis in goats in Switzerland (Pusterla et al., 1997) and in sheep and goats in western Norway ( J. Scho¨nheit and K. Handeland, unpublished). Furthermore, there seem to be no reports of inoculation experiments with E. cervi in domestic ruminants. However, Panin (1964) recorded an unsuccessful attempt to transfer E. panticola from Siberian red deer (Cervus elaphus sibiricus) to sheep. This parasite was considered by Gibbons et al. (1991) to be identical with E. cervi. This paper presents the results of an inoculation experiment undertaken to study the infectivity of E. cervi for sheep and goats.
Materials and Methods Animals, Inocula and Clinical Observations Eight goats (three males, five females) of the Norwegian dairy goat breed were purchased from a herd in Telemark county when 4 months old. The goats had been fed fermented milk replacer, hay and concentrates, and had not been on pasture. Six male sheep of the Old Norwegian breed, originating from a local herd (Oslo county), were taken from their summer pasture when 4 months old. During the experiment all animals were kept indoors and fed hay and concentrates. Six of the goats (goats 1–6) and four of the sheep (sheep 1–4) were given an aqueous suspension of squashed snails containing L3 of E. cervi by stomach tube (Table 1), while two goats and two sheep were kept as uninfected controls. The inoculation of goat 3 had been carried out before the main experiment as a pilot study, and the small inoculum given to this goat (Table 1) was a result of low production of L3 in the snails. The animals were anaesthetized with barbiturates, bled and subjected to necropsy at various times post-inoculation (PI) (Table 1), whereas the controls were killed for examination at the end of the experiment. For infecting intermediate hosts, L1 of E. cervi were obtained from the faeces of free-living red deer culled on the island of Svanøy, western Norway. Other cervid species are absent from Svanøy and the surrounding mainland ( J. T. Solheim, personal communication). The lungs of the red deer were examined grossly and histologically and
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found to contain Elaphostrongylus eggs. Microscopical examination revealed dorsal-spined L1 in the bronchial mucus. Infection with Varestrongylus sagittatus, the protostrongylid lung worm of red deer producing nodular lung lesions and dorsal-spined L1 resembling those of E. cervi (Kutzer and Prosl, 1975), was not found. Snails of the species Arianta arbustorum were exposed to L1 and examined as described by Skorping (1982). The number of L3 given to each experimental animal was estimated on the basis of the L3 average of 2·5 (range 1–6; goat 3) and 32 (range 27–47; goats 1, 2, 4, 5 and 6, and sheep 1–4) found in 10 snails examined at the time of preparing the squashed snail suspension. The animals were observed daily throughout the experiment. Twice a week, faecal samples of 5–10 g were collected from the rectum and examined for L1 of E. cervi after baermann extraction. At each faecal sampling, all animals were observed while walking, and tested for pelvic limb posture reflexes.
Post-mortem Examination At necropsy, the skeletal muscles were searched carefully for the presence of nematodes, with the naked eye and under a strong light. The skull was split in the sagittal plane and the vertebral column was opened from the dorsal surface. The two halves of the brain and the entire spinal cord, including nerve roots, were removed and, together with the cranial cavity and vertebral canal, examined macroscopically for the presence of nematodes. The brain, spinal cord and spinal nerve roots were collected, together with 10–15 samples (either from areas showing gross lesions, or randomly selected from apparently normal areas) from each of the following: lungs, heart, liver and kidneys. All samples were fixed in 10% buffered formalin. Samples of the small intestine, large intestine, abomasum and gastrointestinal lymph nodes were also taken from goats 1 and 2. The lymph node groups examined were the atrial and right ruminal nodes, the dorsal abomasal nodes, the pancreaticoduodenal nodes, the jejunal nodes, the ileocolic nodes, and the colic nodes. After fixation, entire transverse tissue blocks of the brain and spinal cord were cut at 5-mm intervals. One of the blocks from each spinal cord segment included the spinal nerve roots. The lumbosacral spinal nerve roots were removed separately. The CNS tissue blocks, the lumbosacral spinal nerve roots and the other organ samples were embedded in paraffin wax, sectioned at 2–4 m, and stained with haematoxylin and eosin (HE) for histological examination. Selected
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Table 1 Number of gross lesions and nematodes in the CNS of goats and sheep at intervals after inoculation with E. cervi L3 Animal
Goat 1 2 3 4 5 6 Sheep 1 2 3 4
Dose of larvae
Days between inoculation and necropsy
290 290 60 290 160 290 160 160 380 160
6 14 28 49 150 150 21 49 77 143
Number of gross lesions∗ in
nematodes† in
liver
lungs
heart
kidneys
brain
spinal cord
++++ +++ + − − − ++ + + −
++ +++ + + − − ?‡ ?‡ ?‡ ?‡
− + − + − + − − − −
+ +++ + ++++ + + + + ++ +
0 2 0 0 0 0 0 1 2 5
0 6 3 2 0 0 0 1 1 11
∗−, None; +, 1–10; ++, 11–20; +++, 21–30; ++++, 31–40. †Includes histologically identified nematodes, and three nematodes detected macroscopically in sheep 4. ‡The number of E. cervi-induced lesions could not be determined due to the simultaneous presence of lesions caused by Dictyocaulus sp.
CNS sections were also stained with periodic acidSchiff (PAS). During processing of CNS slide preparations there was a considerable loss of meninges, especially in preparations from the brain. Elaphostrongylus nematodes found macroscopically were removed and preserved in 70% ethanol and subsequently identified according to the prevailing description of the species (Steen et al., 1989; Gibbons et al., 1991). All nematode crosssections located on the same slide during histological examination of the CNS were assumed to represent one and the same parasite. Nematode larvae with a cross-sectional diameter similar to that of the Elaphostrongylus L3 stage (Mitskevich, 1964; Panin, 1964) were defined as L3, whereas nematodes containing well developed gonads or genital ducts with sex cells were defined as mature. Nematodes with cross-sections indicating a developmental stage between the L3 and mature stages were defined as L4/L5. Results Clinical Findings No L1 of E. cervi were recovered from any faecal sample. Two goats (nos 5 and 6) and one sheep (no. 4) showed asymmetrical paraparesis and delayed pelvic limb posture reflexes during the period 6 to 18 weeks PI. Subsequently, reduced vision, mental confusion, tetraparesis, right head tilt and lowered right palpebra and ear occurred in the sheep. At 143 days PI, sheep 4 was found in lateral
recumbency, unable to rise and with no appetite. It was therefore humanely destroyed. Gross Findings The carcasses of goats 5 and 6, and particularly that of sheep 4, were thinner than those of the control animals. In the brain of sheep 4, three coiled, hair-thin nematodes, 3–5 cm long, were detected in the subdural (Fig. 1) and subarachnoid spaces of the cerebral hemispheres and in the wall of a lateral brain ventricle. They were identified as mature E. cervi. The presence and number of Elaphostrongylus-associated lesions seen in different organs of the individual experimental animals are summarized in Table 1. Similar lesions were not found in the controls. Small red or greyish foci were seen on the lung surfaces. In the lungs of all sheep (including the controls), focal atelectasis and emphysema were also seen, associated with the presence of a Dictyocaulus sp. On the hearts, there were small greyishyellow foci, and one larger greyish-white area (infarct) that extended throughout the myocardial wall (Fig. 2). The livers showed small greyish-yellow foci, mainly on the diaphragmatic surface, and similar lesions also occurred on the cut surfaces. One small red focus was seen on the kidneys of goat 1. Numerous foci of discoloration and one area of acute infarction were found on the kidney surfaces of goat 2 (Fig. 3). The kidneys of the remaining animals showed small greyish foci and
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Fig. 1. Sheep 4. One coiled E. cervi free on the dura mater of the cranial cavity. Fig. 2. Goat 6. Infarcted area (arrow) extending throughout the myocardial wall of the left ventricle. Fig. 3. Goat 2. One red protruding area of infarction (right) and multiple grey–yellow foci (arrowheads) on the kidney surface.
a few larger, markedly depressed areas with triangular extensions into the renal cortex (old infarcts). Histological Findings Nematodes or associated tissue lesions were not detected histologically in the organs of the controls, except for Dictyocaulus-induced lesions in the lungs. Nervous system. Thirty-one nematodes were identified histologically in the CNS of the infected animals (Table 1). Of these, 25 were located in the subarachnoid spaces (Fig. 4), the remainder occurring in a lateral ventricle (sheep 3), the brain parenchyma (goat 2, sheep 4) (Fig. 5), and the subdural space of the spinal cord (sheep 4). The nematodes seen in goat 2 were L3, whereas those in sheep 4 were all mature. Those in the remaining animals included both mature nematodes and L4/ L5. Two slight accumulations of inflammatory cells in the leptomeninges of the brain were the only nervous system lesions found in goat 1. In all other animals, inflammatory lesions occurred throughout the CNS and in the spinal nerve roots along the
entire spinal cord. The lesions included accumulations of lymphocytes, plasma cells, macrophages and variable numbers of eosinophils, as well as scattered granulomas. The granulomas were lymphohistiocytic, or contained giant cells and, in some sites, remains of nematode material. Leptomeningeal lesions were usually focal and occasionally included thrombosed arterioles. A diffuse leptomeningitis was seen in the brains of the animals killed 49–150 days PI. Dural lesions were located mainly in the dural sheaths of the spinal nerve roots and were most severe in the lumbosacral region. Lesions also occurred in the perineurium of epidural parts of the spinal nerve roots and in the surrounding epidural adipose tissue. Foci of degeneration and necrosis were observed in subarachnoid parts of the nerve root fascicles of the lumbosacral spinal cord (Fig. 6). Cells resembling unsegmented nematode eggs were found in the sub-dural space of the cervical spinal cord of sheep 4. The CNS parenchyma showed perivascular cuffs and scattered foci of gliosis. Numerous malacic foci left by migrating nematodes, and extensive areas of gliosis, were seen in the cerebrum of sheep 4 (Fig. 7). Inflammatory lesions also occurred in the choroid plexus and wall of the brain ventricles (Fig. 8).
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Fig. 4. Sheep 2. Cross-sections of an immature E. cervi female in the subarachnoid space of the cerebellum. HE. ×200. Fig. 5. Sheep 4. Cross-sections of a mature E. cervi male in the cerebrum, within a migratory tunnel filled with erythrocytes. HE. ×100.
Fig. 6. Goat 4. Parasitic granuloma in the subarachnoid space of the sacral spinal cord extending into a nerve root fascicle (N). D, dura mater. HE. ×100. Fig. 7. Sheep 4. One large fresh malacic focus (nematode migratory tract) with compressed margins filled with erythrocytes in the cerebral tissue. Older malacic lesions (arrowheads), diffuse gliosis, and perivascular cuffs are present in the surrounding tissue. HE. ×100.
Lungs. Lung lesions found in goats 1–3 and sheep 1 consisted of focal haemorrhages, necrotic areas and accumulations of eosinophils, neutrophils and
mononuclear inflammatory cells. In some sites the lesions included thrombosed arterioles, giant cells and remains of nematode material; one nematode
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Fig. 8. Goat 5. Granulomas in the wall of a lateral brain ventricle. HE. ×100. Fig. 9. Sheep 1. Inflammation, focal necrosis and cross-section of nematode larva (E. cervi L3) (arrow) in the lung tissue. HE. ×100.
larva with a diameter (44 m) twice that of Dictyocaulus L1 and similar to that of L3 of Elaphostrongylus was sectioned in the lungs of sheep 1 (Fig. 9). In the other animals, there were scattered accumulations of mononuclear cells, lymphohistiocytic granulomas and fibrotic scars. Heart. In goat 2 and sheep 1 there were numerous foci of muscular degeneration and necrosis, and accumulations of eosinophilic and mononuclear inflammatory cells; one thrombosed arteriole was observed within a macroscopic lesion (infarct). In animals killed at later stages, lesions consisted of scattered interstitial accumulations of mononuclear cells and fibrotic scars. Liver. The livers of goats 1 and 2 and sheep 1 contained numerous foci of hepatocellular degeneration and necrosis and accumulations of mainly eosinophilic inflammatory cells in the portal triads. Occasionally, necrotic lesions included thrombosed vessels and remains of nematode material, and one L3 was sectioned (Fig. 10). In the other animals, there were scattered granulomas, foci of accumulated mononuclear cells and proliferated fibroblasts, and fibrotic scars. Kidneys. One L3 was sectioned in the renal cortex of goat 1 (Fig. 11). In the renal cortex of goat 2
and sheep 1 there were many focal lesions consisting of inflammatory cells, extravasated erythrocytes, necrotic and regenerating tubuli, and proliferating fibroblasts. Some of the lesions included thrombosed vessels (infarcts), giant cells and remains of nematode material (granulomas). The remaining animals showed similar lesions in the chronic stage. Gastrointestinal wall and lymph nodes (goats 1 and 2). The abomasum and small intestine of both animals, as well as the large intestine of goat 2, showed focal accumulations, mainly of eosinophilic inflammatory cells, affecting all layers of the wall. In the submucosa of the abomasum, lymphohistiocytic granulomas, thrombosed venules, and one L3 could be demonstrated (Fig. 12). Accumulations of inflammatory cells also occurred close to vessels and nerves in the gastrointestinal serosa and mesenteric tissue. All lymph node groups examined, except for the colic nodes in goat 1, showed accumulations of predominantly eosinophilic inflammatory cells and occasional granulomas (Fig. 13). One L3 was sectioned in a sinus of goat 1. Discussion This study showed that E. cervi can infect and may cause neurological disease in sheep and goats. The
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Fig. 10. Goat 2. Liver tissue with an E. cervi L3 and its migratory tract partly filled with debris and inflammatory cells. HE. ×100. Fig. 11. Goat 1. Cross-section of an E. cervi L3 (arrow) within a dilated capillary in the renal cortex. HE. ×200.
Fig. 12. Goat 1. Granulomas and cross-section of an E. cervi L3 (arrow) in the submucosa of abomasum. HE. ×100. Fig. 13. Goat 1. Granuloma (arrow) in the subcapsular sinus, and inflammation in the capsule and serosa of a gastrointestinal lymph node. HE. ×100.
L3 followed a haematogenous migratory route from the abomasal and small intestinal walls via the porto-hepatic system to the lungs and then spread to all tissues via the general circulation. The findings also indicated that some larval migration to
the lungs occurs via the lymph system. Nematode development took place in the CNS. Haematogenous larval spread to the CNS was suggested for E. cervi in its natural red deer host by Handeland et al. (2000), but they were unable to determine the
E. cervi Infection in Sheep and Goats
site of entry of the L3 into the gastrointestinal wall or the larval migratory route to the lungs. The migratory route of the L3 of E. cervi found in sheep and goats in the present study was the same as that found for E. rangiferi in reindeer and small ruminants (Handeland and Skorping, 1992a; Handeland et al., 1993; Handeland, 1994). The first evidence of haematogenous larval migration of E. cervi to the CNS was found in experimentally infected guinea-pigs (Demiaszkiewicz, 1989). However, in an account of a recent E. cervi study in guinea-pigs (Olsson et al., 1998), it was suggested that the L3 reach the CNS (spinal cord) by direct migration along the spinal nerves. Moreover, the haematogenous migratory route reported for E. rangiferi in small ruminants and reindeer (Handeland and Skorping, 1992a; Handeland, 1994) has been questioned. However, the presence of L3 and numerous necrotic and inflammatory foci, including infarcts, in organs with functional end arteries (kidneys, heart, CNS) in small ruminants in the present study, as well as in the studies on E. rangiferi, is strong evidence of a massive spread of the two parasites via the general circulation. The presence of L3 and tissue lesions in the brain during the early stage of infection in these studies obviously results from haematogenous spread and not from larval migration from the peritoneal cavity. The observations that more nematodes were located in the spinal cord than in the brain, and that inflammatory lesions occurred along the spinal nerve roots of the cord, indicated that haematogenously spread larvae, arrested in arterial vessels outside the cord, migrated into the cord along the spinal nerves. The parallel arrangement of spinal nerves and vessels in the tissues probably makes it easy for the larvae to find nerves to migrate along into the spinal cord. A similar larval migration into the cord by E. cervi seemed to occur in red deer (Handeland et al., 2000), and has been found for E. rangiferi both in reindeer and in small ruminants (Handeland and Skorping, 1992a,b; Handeland, 1994). However, it should not be ruled out (1) that there might also be some direct larval migration from the wall of the gastrointestinal tract via the mesenteric nerves to the CNS, or (2) that larvae which have entered the abdominal cavity might migrate to the CNS via the spinal nerves in the abdominal wall, as proposed in the report by Olsson et al. (1998) of studies in guinea-pigs. The former suggestion was supported by the presence of inflammatory lesions close to the mesenteric nerves in the present study. The main site of development of E. cervi in the
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CNS of sheep and goats was in the subarachnoid spaces. However, developing larvae and mature nematodes also occurred in other CNS locations such as the brain ventricles and brain parenchyma. This was in contrast to findings made in red deer infected experimentally, in which nematodes were confined to the subarachnoid spaces, presumably as a result of migration to that site of larvae that arrived haematogenously in other CNS locations (Handeland et al., 2000). Our findings may indicate that migration of the nematode from other CNS locations to its main site of development in the subarachnoid spaces is less successful in sheep and goats than in its natural red deer host. The same conclusion was reached for E. rangiferi in small ruminants rather than in reindeer (Handeland and Skorping, 1992b; Handeland, 1994). The number of intact nematodes recovered from the CNS in the present study, expressed as a percentage of the number of L3 administrated, was low (about 1%) by comparison with that for E. cervi in experimentally infected red deer (about 6%) (Handeland et al., 2000). The low rate of nematode recovery from the sheep and goats was probably due to mortality of larvae during development. In contrast, death of E. cervi was not confirmed in the red deer. It seems probable that the greater resistance of sheep and goats than red deer to E. cervi not only results in the death of nematodes in the CNS, but also results in less successful invasion of the gastrointestinal mucosa and migration of the L3 to the CNS. The study showed that the ability of E. cervi to reach the adult (mature) stage in small ruminants was poor and that patent infections did not occur. However, in at least one of the animals (sheep 4), the nematode reached the mature stage in sufficient numbers to permit reproduction, and cells resembling unsegmented nematode eggs were observed in the spinal cord. The prepatent period of E. cervi in the natural host is usually 3–4 months (Watson, 1983); it may be longer (up to 206 days), however, in low-grade experimental infections (Gajadhar et al., 1994). It is possible, therefore, that the mature nematodes found in the CNS of sheep 4, killed 143 days PI, would have been capable of producing a patent infection. We conclude that small ruminants are unsuitable hosts for E. cervi. If the parasite succeeded in reproducing in an occasional animal, the excretion of L1 would probably be slight, of short duration, and inadequate for maintaining infection in a small ruminant population. The initial clinical sign in the two goats and one sheep that developed neurological disturbances was
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posterior paresis. This was also an initial sign in guinea-pigs and goats experimentally infected with E. cervi and E. rangiferi, respectively (Watson and Gill, 1985; Demiaszkiewicz, 1989; Handeland and Skorping, 1993). In the present study, posterior paresis was apparently linked to lesions in the subarachnoid spinal nerve roots, caused by nematodes developing in the spinal subarachnoid space. Moreover, the perineural and dural sheath lesions in the extradural spinal nerve roots, also involving the nerve tissue, should be considered a contributing factor. These inflammatory lesions in the extradural parts of the spinal nerves were presumably caused by the migration of L3 towards the cord. The slight signs of posterior paresis were in contrast to the severe brain signs that occurred in sheep 4 from 19 weeks PI. These brain signs were produced by a few adult E. cervi in the brain parenchyma, causing extensive areas of gliosis and focal traumatic malacia. The severe nervous signs that occurred in a goat 3 months after experimental infection with E. rangiferi were also attributable to traumatic lesions in the brain parenchyma caused by migrating adult nematodes (Handeland and Skorping, 1992b, 1993). Moreover, the migration of a few adult nematodes in the brain or spinal cord parenchyma is the main cause of clinical disease in goats and sheep naturally infected with E. rangiferi (Handeland, 1991; Handeland and Sparboe, 1991). A similar pathogenetic mechanism can be expected in natural E. cervi infection in small ruminants. Focal malacia due to nematode migration in the CNS parenchyma was also reported in Swiss goats suspected to be infected by E. cervi (Pusterla et al., 1997). The random migration of adult E. cervi in the brain parenchyma, as found in sheep 4 in this study, as well as of adult E. rangiferi in the brain and spinal cord parenchyma of small ruminants, may have several explanations. It may be associated with nematodes that have developed in the CNS parenchyma following retarded migration to the subarachnoid spaces in these unnatural hosts. Alternatively, the nematodes concerned may have entered the parenchyma during migration from sites of development in the inner cerebrospinal fluid system, such as the brain ventricles and central canal of the cord. In the present study, adult E. cervi were identified within, or in the wall of, the brain ventricles, while adult E. rangiferi have been found both in the brain ventricles and in the central canal of the cord in small ruminants (Handeland and Skorping, 1992b). Irrespective of whether they reach the adult stage in the CNS parenchyma, the
brain ventricles or the central canal of the cord, adult nematodes can be expected to produce laceration of the CNS parenchyma when they start their search for a sexual partner and embark upon their migration towards the skeletal muscles. In the light of the analogous infectivity and pathogenesis of E. cervi and E. rangiferi in small ruminants, we suggest that E. cervi infection is a potential cause of neurological disease in small ruminants grazing in areas inhabited by red deer. Our findings strengthen the belief that E. cervi infection is a plausible explanation for the cerebrospinal nematodosis reported in Swiss goats (Pusterla et al., 1997). After this paper had been submitted for publication, we became aware of a report of neurological signs, death and the presence of larvae in the CNS of two goats experimentally infected with 1000 and 5000 L3 of E. cervi (Demiaszkiewicz et al., 1999). Acknowledgments This study was carried out with the approval of the Norwegian Animal Research Authority. The authors thank J. T. Solheim for sampling lungs and faeces from E. cervi-infected red deer, E. Andersen for the rearing and supply of snails, O. Erdal and K. Witberg for animal management and assistance during clinical sampling and examination, and A. Stovner, E. Bøhn, P. Fagerli and G. Torgersen for technical assistance. We also thank B. Gjerde, I. Bjerkaas and D. Jacobs for critically reviewing the manuscript. References Borg, K. (1979). Symptoms of a lumbar paralysis in a red deer calf on the occasion of Elaphostrongylus infestation of the cerebellum. Zeitschrift fu¨r Jagdwissenschaft, 25, 237–238. Demiaszkiewicz, A. W. (1989). Migration of invasive larvae of Elaphostrongylus cervi Cameron, 1931, and their development to maturity in guinea pigs. Acta Parasitologica, 34, 39–43. Demiaszkiewicz, A. W., Drozdz, J., Lachowicz, J. and Bielecki, W. (1999). Experimental infection of goats with Elaphostrongylus cervi (Nematoda, Protostrongylidae) strain of invasive larvae. Veterinary Bulletin, 69, 1176. English abstract; paper published in Polish, Medycyna Weterynaryjna, 55, 465–467. Gajadhar, A. A., Tessaro, S. V. and Yates, W. D. G. (1994). Diagnosis of Elaphostrongylus cervi infection in New Zealand red deer (Cervus elaphus) quarantined in Canada, and experimental determination of a new extended prepatent period. Canadian Veterinary Journal, 35, 433–437.
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Gibbons, L. M., Halvorsen, O. and Stuve, G. (1991). Revision of the genus Elaphostrongylus Cameron (Nematoda, Metastrongyloidea) with particular reference to species of the genus occurring in Norwegian cervids. Zoologica Scripta, 20, 15–26. Handeland, K. (1991). Cerebrospinal elaphostrongylosis in sheep in northern Norway. Journal of Veterinary Medicine Series B, 38, 773–780. Handeland, K. (1994). Experimental studies of Elaphostrongylus rangiferi in reindeer (Rangifer tarandus tarandus): II. Life cycle, pathogenesis, and pathology. Journal of Veterinary Medicine Series B, 41, 351–365. Handeland, K., Gibbons, L. M. and Skorping, A. (2000). Aspects of the life cycle and pathogenesis of Elaphostrongylus cervi in red deer (Cervus elaphus). Journal of Parasitology, in press. Handeland, K. and Skorping, A. (1992a). The early migration of Elaphostrongylus rangiferi in goats. Journal of Veterinary Medicine Series B, 39, 263–272. Handeland, K. and Skorping, A. (1992b). Experimental cerebrospinal elaphostrongylosis (Elaphostrongylus rangiferi) in goats: II. Pathological findings. Journal of Veterinary Medicine Series B, 39, 713–722. Handeland, K. and Skorping, A. (1993). Experimental cerebrospinal elaphostrongylosis (Elaphostrongylus rangiferi) in goats: I. Clinical observations. Journal of Veterinary Medicine Series B, 40, 141–147. Handeland, K., Skorping, A. and Slettbakk, T. (1993). Experimental cerebrospinal elaphostrongylosis (Elaphostrongylus rangiferi) in sheep. Journal of Veterinary Medicine Series B, 40, 181–189. Handeland, K., Skorping, A., Stuen, S. and Slettbakk, T. (1994). Experimental studies of Elaphostrongylus rangiferi in reindeer (Rangifer tarandus tarandus): Clinical observations. Rangifer, 14, 83–87. Handeland, K. and Slettbakk, T. (1995). Epidemiological aspects of cerebrospinal elaphostrongylosis in goats and sheep in northern Norway. Journal of Veterinary Medicine Series B, 42, 110– 117. Handeland, K. and Sparboe, O. (1991). Cerebrospinal elaphostrongylosis in dairy goats in northern Norway. Journal of Veterinary Medicine Series B, 38, 755–763. Hemmingsen, W., Halvorsen, O. and Skorping, A. (1993). Migration of adult Elaphostrongylus rangiferi (Nematoda: Protostrongylidae) from the spinal subdural space to the muscles of reindeer (Rangifer tarandus). Journal of Parasitology, 79, 728–732. Kutzer, E. and Prosl, H. (1975). Contribution to the knowledge of Elaphostrongylus cervi Cameron, 1931. I. Morphology and diagnosis. Wiener Tiera¨rztliche Monatsschrift, 62, 258–266.
Mitskevich, V. Y. (1964). Life cycle of Elaphostrongylus rangiferi MIZ. 1958. In: Parasites of Farm Animals in Kazakhstan, S. N. Boev, Ed., Izdatel’stvo Akademii Nauk Kazakhstan SSR, Alma-Ata, pp. 49–60. Translated from Russian by the Israel Program for Scientific Translations, National Science Foundation, Washington, 1967, IPST catalogue number 1868. Olsson, I. M., Lankester, M. W., Gajadhar, A. A. and Steen, M. (1998). Tissue migration of Elaphostrongylus spp. in guinea pigs (Cavia porcellus). Journal of Parasitology, 84, 968–975. Panin, V. Y. (1964). Life cycle of Elaphostrongylus panticola Lubimov, 1945. In: Parasites of Farm Animals in Kazakhstan, S. N. Boev, Ed., Izdatel’stvo Akademii Nauk Kazakhstan SSR, Alma-Ata, pp. 34–48. Translated from Russian by the Israel Program for Scientific Translations, National Science Foundation, Washington, 1967, IPST catalogue number 1868. Prosl, H. and Kutzer, E. (1980). The pathology of Elaphostrongylus infection in red deer. Monatshefte fu¨r Veterina¨rmedizin, 35, 151–153. Pusterla, N. P., Caplazi, P. and Braun, U. (1997). Cerebrospinal nematodiasis in seven goats. Schweizer Archiv fu¨r Tierheilkunde, 139, 282–287. Roneus, O. and Nordkvist, M. (1962). Cerebrospinal and muscular nematodiasis (Elaphostrongylus rangiferi) in Swedish reindeer. Acta Veterinaria Scandinavica, 3, 201–225. Skorping, A. (1982). Elaphostrongylus rangiferi: Influence of temperature, substrate, and larval age on the infection rate in the intermediate snail host, Arianta arbustorum. Experimental Parasitology, 54, 222–228. Skorping, A. and Halvorsen, O. (1980). The susceptibility of terrestrial gastropods to experimental infection with Elaphostrongylus rangiferi Mitskevich (Nematoda: Metastrongyloidea). Zeitschrift fu¨r Parasitenkunde, 62, 7–14. Steen, M., Chabaud, A. G. and Rehbinder, C. (1989). Species of the genus Elaphostrongylus parasite of Swedish cervidae. A description of E. alces n. sp. Annales de Parasitologie Humaine et Compare´e, 64, 134–142. Watson, T. G. (1983). Some clinical and parasitological features of Elaphostrongylus cervi infections in Cervus elaphus. New Zealand Journal of Zoology, 10, 129. Watson, T. G. and Gill, J. M. (1985). The experimental infection of guinea pigs with the tissue worm of deer—Elaphostrongylus cervi. New Zealand Veterinary Journal, 33, 81–83.
Received, November 8th, 1999 Accepted, April 18th, 2000
Experimental Elaphostrongylus cervi Infection in Sheep and Goats K. Handeland, L. M. Gibbons∗ and A. Skorping† Section of Wildlife Diseases, National Veterinary Institute, P.O. Box 8156 Dep., N-0033 Oslo, Norway, ∗Department of Pathology and Infectious Diseases, The Royal Veterinary College, University of London, Hawkshead Lane, North Mymms, Hatfield, Herts AL9 7TA, UK and †Department of Zoology, University of Bergen, Allegt. 41, 5007 Bergen, Norway Summary The pathogenesis and migratory life cycle of Elaphostrongylus cervi were studied in four sheep and six goats killed and examined 6 days to 5 months after inoculation with infective third-stage larvae (L3). Detailed histological studies demonstrated that the L3 followed a porto-hepatic, and probably also a secondary lymphatic, migratory route from the abomasum and small intestine to the lungs, with subsequent spread via the general circulation to the central nervous system (CNS) and other tissues. In addition, the results suggested that haematogenously spread L3, arrested in arterial vessels outside the spinal cord, migrated into the cord along the spinal nerves. During migration, the L3 caused focal inflammation and necrosis in the organs and along the spinal nerve roots, and infarcts occurred in the myocardium, kidneys and CNS. Nematode development took place in the CNS. During development, there was a gradual die-off of nematodes and patent infections were not observed. However, in one animal many mature nematodes were demonstrated in the CNS. In the nervous system, the nematodes caused encephalomyelitis, focal traumatic encephalomalacia, gliosis, meningitis, choroiditis, radiculitis and perineuritis. Two goats and one sheep displayed long-lasting paraparesis starting 6 weeks after inoculation. The signs apparently resulted from nematode-induced spinal nerve root lesions. From 19 weeks after inoculation the sheep also showed signs of severe brain disturbances due to traumatic and inflammatory lesions caused by adult E. cervi in the cerebral parenchyma. We conclude that E. cervi represents a potential cause of neurological disease in small ruminants grazing areas inhabited by red deer. This is the first report confirming the infectivity of E. cervi for domestic ruminants. 2000 Harcourt Publishers Ltd
Introduction In their definitive hosts the protostrongylid nematodes Elaphostrongylus rangiferi of reindeer (Rangifer tarandus) and E. cervi of red deer (Cervus elaphus) develop to the adult stage in the central nervous system (CNS) and then migrate to the skeletal muscles (Hemmingsen et al., 1993; Handeland, 1994; Handeland et al., 2000). The hosts may develop neurological signs during the CNS phase of the infection (Roneus and Nordkvist, 1962; Borg, 1979; Handeland et al., 1994). Mature females deposit eggs into veins and the eggs are carried haematogenously to the lungs (Prosl and Kutzer, 1980). Patent infections are characterized by the occurrence of nematode eggs disseminated throughout the lung tissue (Prosl and Kutzer, 1980) 0021–9975/00/080248+10 $35.00
and the presence of dorsal-spined first-stage larvae (L1) in the bronchial mucus and faeces (Mitskevich, 1964). The free-living L1 develop to the infective third-stage (L3) in various intermediate snail hosts (Skorping and Halvorsen, 1980) with definitive hosts becoming infected through accidental ingestion of snails containing L3, mainly during late summer (Mitskevich, 1964; Panin, 1964). Elaphostrongylus rangiferi is known to infect sheep and goats grazing in areas with reindeer, causing neurological disease (Handeland, 1991; Handeland and Sparboe, 1991; Handeland and Slettbakk, 1995). Signs are normally associated with the migration of adult nematodes in the CNS parenchyma. Experimental studies have shown that there is a gradual die-off of the parasite during its development in the CNS of small ruminants and 2000 Harcourt Publishers Ltd
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the parasite does not seem to reach patency in these unnatural hosts (Handeland and Skorping, 1992b, 1993; Handeland et al., 1993). Although infection with E. cervi has not been confirmed in domestic livestock, this nematode is considered a plausible cause of cerebrospinal nematodosis in goats in Switzerland (Pusterla et al., 1997) and in sheep and goats in western Norway ( J. Scho¨nheit and K. Handeland, unpublished). Furthermore, there seem to be no reports of inoculation experiments with E. cervi in domestic ruminants. However, Panin (1964) recorded an unsuccessful attempt to transfer E. panticola from Siberian red deer (Cervus elaphus sibiricus) to sheep. This parasite was considered by Gibbons et al. (1991) to be identical with E. cervi. This paper presents the results of an inoculation experiment undertaken to study the infectivity of E. cervi for sheep and goats.
Materials and Methods Animals, Inocula and Clinical Observations Eight goats (three males, five females) of the Norwegian dairy goat breed were purchased from a herd in Telemark county when 4 months old. The goats had been fed fermented milk replacer, hay and concentrates, and had not been on pasture. Six male sheep of the Old Norwegian breed, originating from a local herd (Oslo county), were taken from their summer pasture when 4 months old. During the experiment all animals were kept indoors and fed hay and concentrates. Six of the goats (goats 1–6) and four of the sheep (sheep 1–4) were given an aqueous suspension of squashed snails containing L3 of E. cervi by stomach tube (Table 1), while two goats and two sheep were kept as uninfected controls. The inoculation of goat 3 had been carried out before the main experiment as a pilot study, and the small inoculum given to this goat (Table 1) was a result of low production of L3 in the snails. The animals were anaesthetized with barbiturates, bled and subjected to necropsy at various times post-inoculation (PI) (Table 1), whereas the controls were killed for examination at the end of the experiment. For infecting intermediate hosts, L1 of E. cervi were obtained from the faeces of free-living red deer culled on the island of Svanøy, western Norway. Other cervid species are absent from Svanøy and the surrounding mainland ( J. T. Solheim, personal communication). The lungs of the red deer were examined grossly and histologically and
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found to contain Elaphostrongylus eggs. Microscopical examination revealed dorsal-spined L1 in the bronchial mucus. Infection with Varestrongylus sagittatus, the protostrongylid lung worm of red deer producing nodular lung lesions and dorsal-spined L1 resembling those of E. cervi (Kutzer and Prosl, 1975), was not found. Snails of the species Arianta arbustorum were exposed to L1 and examined as described by Skorping (1982). The number of L3 given to each experimental animal was estimated on the basis of the L3 average of 2·5 (range 1–6; goat 3) and 32 (range 27–47; goats 1, 2, 4, 5 and 6, and sheep 1–4) found in 10 snails examined at the time of preparing the squashed snail suspension. The animals were observed daily throughout the experiment. Twice a week, faecal samples of 5–10 g were collected from the rectum and examined for L1 of E. cervi after baermann extraction. At each faecal sampling, all animals were observed while walking, and tested for pelvic limb posture reflexes.
Post-mortem Examination At necropsy, the skeletal muscles were searched carefully for the presence of nematodes, with the naked eye and under a strong light. The skull was split in the sagittal plane and the vertebral column was opened from the dorsal surface. The two halves of the brain and the entire spinal cord, including nerve roots, were removed and, together with the cranial cavity and vertebral canal, examined macroscopically for the presence of nematodes. The brain, spinal cord and spinal nerve roots were collected, together with 10–15 samples (either from areas showing gross lesions, or randomly selected from apparently normal areas) from each of the following: lungs, heart, liver and kidneys. All samples were fixed in 10% buffered formalin. Samples of the small intestine, large intestine, abomasum and gastrointestinal lymph nodes were also taken from goats 1 and 2. The lymph node groups examined were the atrial and right ruminal nodes, the dorsal abomasal nodes, the pancreaticoduodenal nodes, the jejunal nodes, the ileocolic nodes, and the colic nodes. After fixation, entire transverse tissue blocks of the brain and spinal cord were cut at 5-mm intervals. One of the blocks from each spinal cord segment included the spinal nerve roots. The lumbosacral spinal nerve roots were removed separately. The CNS tissue blocks, the lumbosacral spinal nerve roots and the other organ samples were embedded in paraffin wax, sectioned at 2–4 m, and stained with haematoxylin and eosin (HE) for histological examination. Selected
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Table 1 Number of gross lesions and nematodes in the CNS of goats and sheep at intervals after inoculation with E. cervi L3 Animal
Goat 1 2 3 4 5 6 Sheep 1 2 3 4
Dose of larvae
Days between inoculation and necropsy
290 290 60 290 160 290 160 160 380 160
6 14 28 49 150 150 21 49 77 143
Number of gross lesions∗ in
nematodes† in
liver
lungs
heart
kidneys
brain
spinal cord
++++ +++ + − − − ++ + + −
++ +++ + + − − ?‡ ?‡ ?‡ ?‡
− + − + − + − − − −
+ +++ + ++++ + + + + ++ +
0 2 0 0 0 0 0 1 2 5
0 6 3 2 0 0 0 1 1 11
∗−, None; +, 1–10; ++, 11–20; +++, 21–30; ++++, 31–40. †Includes histologically identified nematodes, and three nematodes detected macroscopically in sheep 4. ‡The number of E. cervi-induced lesions could not be determined due to the simultaneous presence of lesions caused by Dictyocaulus sp.
CNS sections were also stained with periodic acidSchiff (PAS). During processing of CNS slide preparations there was a considerable loss of meninges, especially in preparations from the brain. Elaphostrongylus nematodes found macroscopically were removed and preserved in 70% ethanol and subsequently identified according to the prevailing description of the species (Steen et al., 1989; Gibbons et al., 1991). All nematode crosssections located on the same slide during histological examination of the CNS were assumed to represent one and the same parasite. Nematode larvae with a cross-sectional diameter similar to that of the Elaphostrongylus L3 stage (Mitskevich, 1964; Panin, 1964) were defined as L3, whereas nematodes containing well developed gonads or genital ducts with sex cells were defined as mature. Nematodes with cross-sections indicating a developmental stage between the L3 and mature stages were defined as L4/L5. Results Clinical Findings No L1 of E. cervi were recovered from any faecal sample. Two goats (nos 5 and 6) and one sheep (no. 4) showed asymmetrical paraparesis and delayed pelvic limb posture reflexes during the period 6 to 18 weeks PI. Subsequently, reduced vision, mental confusion, tetraparesis, right head tilt and lowered right palpebra and ear occurred in the sheep. At 143 days PI, sheep 4 was found in lateral
recumbency, unable to rise and with no appetite. It was therefore humanely destroyed. Gross Findings The carcasses of goats 5 and 6, and particularly that of sheep 4, were thinner than those of the control animals. In the brain of sheep 4, three coiled, hair-thin nematodes, 3–5 cm long, were detected in the subdural (Fig. 1) and subarachnoid spaces of the cerebral hemispheres and in the wall of a lateral brain ventricle. They were identified as mature E. cervi. The presence and number of Elaphostrongylus-associated lesions seen in different organs of the individual experimental animals are summarized in Table 1. Similar lesions were not found in the controls. Small red or greyish foci were seen on the lung surfaces. In the lungs of all sheep (including the controls), focal atelectasis and emphysema were also seen, associated with the presence of a Dictyocaulus sp. On the hearts, there were small greyishyellow foci, and one larger greyish-white area (infarct) that extended throughout the myocardial wall (Fig. 2). The livers showed small greyish-yellow foci, mainly on the diaphragmatic surface, and similar lesions also occurred on the cut surfaces. One small red focus was seen on the kidneys of goat 1. Numerous foci of discoloration and one area of acute infarction were found on the kidney surfaces of goat 2 (Fig. 3). The kidneys of the remaining animals showed small greyish foci and
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Fig. 1. Sheep 4. One coiled E. cervi free on the dura mater of the cranial cavity. Fig. 2. Goat 6. Infarcted area (arrow) extending throughout the myocardial wall of the left ventricle. Fig. 3. Goat 2. One red protruding area of infarction (right) and multiple grey–yellow foci (arrowheads) on the kidney surface.
a few larger, markedly depressed areas with triangular extensions into the renal cortex (old infarcts). Histological Findings Nematodes or associated tissue lesions were not detected histologically in the organs of the controls, except for Dictyocaulus-induced lesions in the lungs. Nervous system. Thirty-one nematodes were identified histologically in the CNS of the infected animals (Table 1). Of these, 25 were located in the subarachnoid spaces (Fig. 4), the remainder occurring in a lateral ventricle (sheep 3), the brain parenchyma (goat 2, sheep 4) (Fig. 5), and the subdural space of the spinal cord (sheep 4). The nematodes seen in goat 2 were L3, whereas those in sheep 4 were all mature. Those in the remaining animals included both mature nematodes and L4/ L5. Two slight accumulations of inflammatory cells in the leptomeninges of the brain were the only nervous system lesions found in goat 1. In all other animals, inflammatory lesions occurred throughout the CNS and in the spinal nerve roots along the
entire spinal cord. The lesions included accumulations of lymphocytes, plasma cells, macrophages and variable numbers of eosinophils, as well as scattered granulomas. The granulomas were lymphohistiocytic, or contained giant cells and, in some sites, remains of nematode material. Leptomeningeal lesions were usually focal and occasionally included thrombosed arterioles. A diffuse leptomeningitis was seen in the brains of the animals killed 49–150 days PI. Dural lesions were located mainly in the dural sheaths of the spinal nerve roots and were most severe in the lumbosacral region. Lesions also occurred in the perineurium of epidural parts of the spinal nerve roots and in the surrounding epidural adipose tissue. Foci of degeneration and necrosis were observed in subarachnoid parts of the nerve root fascicles of the lumbosacral spinal cord (Fig. 6). Cells resembling unsegmented nematode eggs were found in the sub-dural space of the cervical spinal cord of sheep 4. The CNS parenchyma showed perivascular cuffs and scattered foci of gliosis. Numerous malacic foci left by migrating nematodes, and extensive areas of gliosis, were seen in the cerebrum of sheep 4 (Fig. 7). Inflammatory lesions also occurred in the choroid plexus and wall of the brain ventricles (Fig. 8).
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Fig. 4. Sheep 2. Cross-sections of an immature E. cervi female in the subarachnoid space of the cerebellum. HE. ×200. Fig. 5. Sheep 4. Cross-sections of a mature E. cervi male in the cerebrum, within a migratory tunnel filled with erythrocytes. HE. ×100.
Fig. 6. Goat 4. Parasitic granuloma in the subarachnoid space of the sacral spinal cord extending into a nerve root fascicle (N). D, dura mater. HE. ×100. Fig. 7. Sheep 4. One large fresh malacic focus (nematode migratory tract) with compressed margins filled with erythrocytes in the cerebral tissue. Older malacic lesions (arrowheads), diffuse gliosis, and perivascular cuffs are present in the surrounding tissue. HE. ×100.
Lungs. Lung lesions found in goats 1–3 and sheep 1 consisted of focal haemorrhages, necrotic areas and accumulations of eosinophils, neutrophils and
mononuclear inflammatory cells. In some sites the lesions included thrombosed arterioles, giant cells and remains of nematode material; one nematode
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Fig. 8. Goat 5. Granulomas in the wall of a lateral brain ventricle. HE. ×100. Fig. 9. Sheep 1. Inflammation, focal necrosis and cross-section of nematode larva (E. cervi L3) (arrow) in the lung tissue. HE. ×100.
larva with a diameter (44 m) twice that of Dictyocaulus L1 and similar to that of L3 of Elaphostrongylus was sectioned in the lungs of sheep 1 (Fig. 9). In the other animals, there were scattered accumulations of mononuclear cells, lymphohistiocytic granulomas and fibrotic scars. Heart. In goat 2 and sheep 1 there were numerous foci of muscular degeneration and necrosis, and accumulations of eosinophilic and mononuclear inflammatory cells; one thrombosed arteriole was observed within a macroscopic lesion (infarct). In animals killed at later stages, lesions consisted of scattered interstitial accumulations of mononuclear cells and fibrotic scars. Liver. The livers of goats 1 and 2 and sheep 1 contained numerous foci of hepatocellular degeneration and necrosis and accumulations of mainly eosinophilic inflammatory cells in the portal triads. Occasionally, necrotic lesions included thrombosed vessels and remains of nematode material, and one L3 was sectioned (Fig. 10). In the other animals, there were scattered granulomas, foci of accumulated mononuclear cells and proliferated fibroblasts, and fibrotic scars. Kidneys. One L3 was sectioned in the renal cortex of goat 1 (Fig. 11). In the renal cortex of goat 2
and sheep 1 there were many focal lesions consisting of inflammatory cells, extravasated erythrocytes, necrotic and regenerating tubuli, and proliferating fibroblasts. Some of the lesions included thrombosed vessels (infarcts), giant cells and remains of nematode material (granulomas). The remaining animals showed similar lesions in the chronic stage. Gastrointestinal wall and lymph nodes (goats 1 and 2). The abomasum and small intestine of both animals, as well as the large intestine of goat 2, showed focal accumulations, mainly of eosinophilic inflammatory cells, affecting all layers of the wall. In the submucosa of the abomasum, lymphohistiocytic granulomas, thrombosed venules, and one L3 could be demonstrated (Fig. 12). Accumulations of inflammatory cells also occurred close to vessels and nerves in the gastrointestinal serosa and mesenteric tissue. All lymph node groups examined, except for the colic nodes in goat 1, showed accumulations of predominantly eosinophilic inflammatory cells and occasional granulomas (Fig. 13). One L3 was sectioned in a sinus of goat 1. Discussion This study showed that E. cervi can infect and may cause neurological disease in sheep and goats. The
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Fig. 10. Goat 2. Liver tissue with an E. cervi L3 and its migratory tract partly filled with debris and inflammatory cells. HE. ×100. Fig. 11. Goat 1. Cross-section of an E. cervi L3 (arrow) within a dilated capillary in the renal cortex. HE. ×200.
Fig. 12. Goat 1. Granulomas and cross-section of an E. cervi L3 (arrow) in the submucosa of abomasum. HE. ×100. Fig. 13. Goat 1. Granuloma (arrow) in the subcapsular sinus, and inflammation in the capsule and serosa of a gastrointestinal lymph node. HE. ×100.
L3 followed a haematogenous migratory route from the abomasal and small intestinal walls via the porto-hepatic system to the lungs and then spread to all tissues via the general circulation. The findings also indicated that some larval migration to
the lungs occurs via the lymph system. Nematode development took place in the CNS. Haematogenous larval spread to the CNS was suggested for E. cervi in its natural red deer host by Handeland et al. (2000), but they were unable to determine the
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site of entry of the L3 into the gastrointestinal wall or the larval migratory route to the lungs. The migratory route of the L3 of E. cervi found in sheep and goats in the present study was the same as that found for E. rangiferi in reindeer and small ruminants (Handeland and Skorping, 1992a; Handeland et al., 1993; Handeland, 1994). The first evidence of haematogenous larval migration of E. cervi to the CNS was found in experimentally infected guinea-pigs (Demiaszkiewicz, 1989). However, in an account of a recent E. cervi study in guinea-pigs (Olsson et al., 1998), it was suggested that the L3 reach the CNS (spinal cord) by direct migration along the spinal nerves. Moreover, the haematogenous migratory route reported for E. rangiferi in small ruminants and reindeer (Handeland and Skorping, 1992a; Handeland, 1994) has been questioned. However, the presence of L3 and numerous necrotic and inflammatory foci, including infarcts, in organs with functional end arteries (kidneys, heart, CNS) in small ruminants in the present study, as well as in the studies on E. rangiferi, is strong evidence of a massive spread of the two parasites via the general circulation. The presence of L3 and tissue lesions in the brain during the early stage of infection in these studies obviously results from haematogenous spread and not from larval migration from the peritoneal cavity. The observations that more nematodes were located in the spinal cord than in the brain, and that inflammatory lesions occurred along the spinal nerve roots of the cord, indicated that haematogenously spread larvae, arrested in arterial vessels outside the cord, migrated into the cord along the spinal nerves. The parallel arrangement of spinal nerves and vessels in the tissues probably makes it easy for the larvae to find nerves to migrate along into the spinal cord. A similar larval migration into the cord by E. cervi seemed to occur in red deer (Handeland et al., 2000), and has been found for E. rangiferi both in reindeer and in small ruminants (Handeland and Skorping, 1992a,b; Handeland, 1994). However, it should not be ruled out (1) that there might also be some direct larval migration from the wall of the gastrointestinal tract via the mesenteric nerves to the CNS, or (2) that larvae which have entered the abdominal cavity might migrate to the CNS via the spinal nerves in the abdominal wall, as proposed in the report by Olsson et al. (1998) of studies in guinea-pigs. The former suggestion was supported by the presence of inflammatory lesions close to the mesenteric nerves in the present study. The main site of development of E. cervi in the
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CNS of sheep and goats was in the subarachnoid spaces. However, developing larvae and mature nematodes also occurred in other CNS locations such as the brain ventricles and brain parenchyma. This was in contrast to findings made in red deer infected experimentally, in which nematodes were confined to the subarachnoid spaces, presumably as a result of migration to that site of larvae that arrived haematogenously in other CNS locations (Handeland et al., 2000). Our findings may indicate that migration of the nematode from other CNS locations to its main site of development in the subarachnoid spaces is less successful in sheep and goats than in its natural red deer host. The same conclusion was reached for E. rangiferi in small ruminants rather than in reindeer (Handeland and Skorping, 1992b; Handeland, 1994). The number of intact nematodes recovered from the CNS in the present study, expressed as a percentage of the number of L3 administrated, was low (about 1%) by comparison with that for E. cervi in experimentally infected red deer (about 6%) (Handeland et al., 2000). The low rate of nematode recovery from the sheep and goats was probably due to mortality of larvae during development. In contrast, death of E. cervi was not confirmed in the red deer. It seems probable that the greater resistance of sheep and goats than red deer to E. cervi not only results in the death of nematodes in the CNS, but also results in less successful invasion of the gastrointestinal mucosa and migration of the L3 to the CNS. The study showed that the ability of E. cervi to reach the adult (mature) stage in small ruminants was poor and that patent infections did not occur. However, in at least one of the animals (sheep 4), the nematode reached the mature stage in sufficient numbers to permit reproduction, and cells resembling unsegmented nematode eggs were observed in the spinal cord. The prepatent period of E. cervi in the natural host is usually 3–4 months (Watson, 1983); it may be longer (up to 206 days), however, in low-grade experimental infections (Gajadhar et al., 1994). It is possible, therefore, that the mature nematodes found in the CNS of sheep 4, killed 143 days PI, would have been capable of producing a patent infection. We conclude that small ruminants are unsuitable hosts for E. cervi. If the parasite succeeded in reproducing in an occasional animal, the excretion of L1 would probably be slight, of short duration, and inadequate for maintaining infection in a small ruminant population. The initial clinical sign in the two goats and one sheep that developed neurological disturbances was
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posterior paresis. This was also an initial sign in guinea-pigs and goats experimentally infected with E. cervi and E. rangiferi, respectively (Watson and Gill, 1985; Demiaszkiewicz, 1989; Handeland and Skorping, 1993). In the present study, posterior paresis was apparently linked to lesions in the subarachnoid spinal nerve roots, caused by nematodes developing in the spinal subarachnoid space. Moreover, the perineural and dural sheath lesions in the extradural spinal nerve roots, also involving the nerve tissue, should be considered a contributing factor. These inflammatory lesions in the extradural parts of the spinal nerves were presumably caused by the migration of L3 towards the cord. The slight signs of posterior paresis were in contrast to the severe brain signs that occurred in sheep 4 from 19 weeks PI. These brain signs were produced by a few adult E. cervi in the brain parenchyma, causing extensive areas of gliosis and focal traumatic malacia. The severe nervous signs that occurred in a goat 3 months after experimental infection with E. rangiferi were also attributable to traumatic lesions in the brain parenchyma caused by migrating adult nematodes (Handeland and Skorping, 1992b, 1993). Moreover, the migration of a few adult nematodes in the brain or spinal cord parenchyma is the main cause of clinical disease in goats and sheep naturally infected with E. rangiferi (Handeland, 1991; Handeland and Sparboe, 1991). A similar pathogenetic mechanism can be expected in natural E. cervi infection in small ruminants. Focal malacia due to nematode migration in the CNS parenchyma was also reported in Swiss goats suspected to be infected by E. cervi (Pusterla et al., 1997). The random migration of adult E. cervi in the brain parenchyma, as found in sheep 4 in this study, as well as of adult E. rangiferi in the brain and spinal cord parenchyma of small ruminants, may have several explanations. It may be associated with nematodes that have developed in the CNS parenchyma following retarded migration to the subarachnoid spaces in these unnatural hosts. Alternatively, the nematodes concerned may have entered the parenchyma during migration from sites of development in the inner cerebrospinal fluid system, such as the brain ventricles and central canal of the cord. In the present study, adult E. cervi were identified within, or in the wall of, the brain ventricles, while adult E. rangiferi have been found both in the brain ventricles and in the central canal of the cord in small ruminants (Handeland and Skorping, 1992b). Irrespective of whether they reach the adult stage in the CNS parenchyma, the
brain ventricles or the central canal of the cord, adult nematodes can be expected to produce laceration of the CNS parenchyma when they start their search for a sexual partner and embark upon their migration towards the skeletal muscles. In the light of the analogous infectivity and pathogenesis of E. cervi and E. rangiferi in small ruminants, we suggest that E. cervi infection is a potential cause of neurological disease in small ruminants grazing in areas inhabited by red deer. Our findings strengthen the belief that E. cervi infection is a plausible explanation for the cerebrospinal nematodosis reported in Swiss goats (Pusterla et al., 1997). After this paper had been submitted for publication, we became aware of a report of neurological signs, death and the presence of larvae in the CNS of two goats experimentally infected with 1000 and 5000 L3 of E. cervi (Demiaszkiewicz et al., 1999). Acknowledgments This study was carried out with the approval of the Norwegian Animal Research Authority. The authors thank J. T. Solheim for sampling lungs and faeces from E. cervi-infected red deer, E. Andersen for the rearing and supply of snails, O. Erdal and K. Witberg for animal management and assistance during clinical sampling and examination, and A. Stovner, E. Bøhn, P. Fagerli and G. Torgersen for technical assistance. We also thank B. Gjerde, I. Bjerkaas and D. Jacobs for critically reviewing the manuscript. References Borg, K. (1979). Symptoms of a lumbar paralysis in a red deer calf on the occasion of Elaphostrongylus infestation of the cerebellum. Zeitschrift fu¨r Jagdwissenschaft, 25, 237–238. Demiaszkiewicz, A. W. (1989). Migration of invasive larvae of Elaphostrongylus cervi Cameron, 1931, and their development to maturity in guinea pigs. Acta Parasitologica, 34, 39–43. Demiaszkiewicz, A. W., Drozdz, J., Lachowicz, J. and Bielecki, W. (1999). Experimental infection of goats with Elaphostrongylus cervi (Nematoda, Protostrongylidae) strain of invasive larvae. Veterinary Bulletin, 69, 1176. English abstract; paper published in Polish, Medycyna Weterynaryjna, 55, 465–467. Gajadhar, A. A., Tessaro, S. V. and Yates, W. D. G. (1994). Diagnosis of Elaphostrongylus cervi infection in New Zealand red deer (Cervus elaphus) quarantined in Canada, and experimental determination of a new extended prepatent period. Canadian Veterinary Journal, 35, 433–437.
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Received, November 8th, 1999 Accepted, April 18th, 2000